Temporal regulation of BMP2 growth factor signaling in response to mechanical loading is linked to cytoskeletal and focal adhesion remodeling

Biophysical cues have the ability to enhance cellular signaling response to Bone Morphogenetic Proteins, an essential growth factor during bone development and regeneration. Yet, therapeutic application of Bone Morphogenetic Protein 2 (BMP2) is restricted due to uncontrolled side effects. An understanding of the temporal characteristics of mechanically regulated signaling events and underlying mechanism is lacking. Using a 3D bioreactor system in combination with a soft macroporous biomaterial substrate, we mimic the in vivo environment that BMP2 is acting in. We show that the intensity and duration of BMP2 signaling increases with increasing loading frequency in synchrony with the number and size of focal adhesions. Long-term mechanical stimulation increases the expression of BMP receptor type 1B, specific integrin subtypes and integrin clustering. Together, this triggered a short-lived mechanical echo that enhanced BMP2 signaling even when BMP2 is administered directly after mechanical stimulation, but not when it is applied after a resting period of ≥30 min. Interfering with cytoskeletal remodeling hinders focal adhesion remodeling verifying its critical role in shifting cells into a state of high BMP2 responsiveness. The design of biomaterials that exploit this potential locally at the site of injury will help to overcome current limitations of clinical growth factor treatment.

1.It would be helpful to explain the full form of BMP-2 and its significance at the beginning of the abstract.
2. Here the authors have provided the expression levels of integrins during differential loading and compression+BMP2 simulation (B/L) (Figure 2F).It would be helpful if the authors can provide fixed sample images showing the distribution of integrins and their clustering at the focal adhesion.This can give a robust insight into integrin clustering, focal adhesion size and actin remodelling.
3. The manuscript is well written generally, but there are a few typos that should be corrected.For example, please rephrase the sentence (line 47-48) "Bone Morphogenetic Protein 2 (BMP-2) is known to play a central role in bone development and regeneration and has gained Besides its indispensable…".Some sentences in the manuscript can also be revisited for easier understanding.For example, the sentence "We wanted to further understand how BMP signalling responds when the time of pre-stimulation is shortened so that cells do not have time to fully adapt to the altered mechanics at all levels, and thereby find out which cellular adaptation processes are responsible for the mechanical echo and the subsequent rapid decay of the crosstalk between mechano-transduction and BMP signalling" can be simplified to gain clarity.
Reviewer #3 (Remarks to the Author) The manuscript by Gorlitz et al describes the aspects of the BMP signalling pathway in the context of how cells in a 3D mechanical bioreactor system respond to a designed temporal stimulation of BMP2 and mechanical compression.The mechano-bioreactor design the authors use allows them to bridge the gap between in vivo bone regeneration and in vitro parameter control.In particular the temporal control of compression and BMP addition.Changes in transcript expression levels of a host of proteins that are (in)directly involved in the BMP pathway were measured as well as the first downstream effect in the BMP pathway: SMAD phosphorylation.Measuring the direct downstream effect of BMP signalling (phosphorylation of SMAD) the authors find that mechanical stimulation augments BMP signalling.This effect is rather immediate (Fig 2A -D, and earlier studies from the authors) and long-term mechanical stimulation also increases negative regulators downstream of SMAD (Fig 4C).These data seem to suggest that the feedforward memory for BMP stimulation is much shorter-lived than feedback memory of the system.To understand the relation between dynamic F-actin remodelling and the augmented BMP signalling upon mechanical stimulation the authors stabilize the actin using Jasplakinolide.There was a complete loss the SMAD phosphorylation increase nor was there a significant increase in the downstream ID1 transcript expression level when BMP signalling was combined with mechanical stimulation in the perturbed situation (Fig 6E).
In general the manuscript is well written but as a whole it is a bit confusing and appears to consist out of two sets of data.One displaying a message of the temporal (dis-)entanglement of the mechanical stimulus and BMP2 signalling (Fig 2, 4 and  5) and a second discussing focal adhesions (Fig 3 and 6).Their relation is not entirely clear.Focal adhesions are as expected bigger in situations where forces are applied to cells (one of many examples is Riveline J Cell Biol 2001).Disrupting actin turnover using Jas directly affects loading-related focal adhesion growth (Galbraith Science 2007, Parsons Nat Rev Mol Cell Biol 2010).Even though the authors subsequently show that Jas perturbation also disrupts loading-related downstream BMP signalling this does not warrant a link with focal adhesion maturation (as mentioned in the title).Perhaps the authors in this regards could make use of the benefits of their system: they could analyse/correlate single cell SMAD nuclear localisation (or phosphorylation) with number/size distribution of focal adhesions.Some more specific comments: 1-The authors find that co-stimulation of BMP and cyclic compression enhances BMP signalling.Using 3 frequencies they find a frequency dependence: 0.03 Hz showed an increased p-SMAD in the first 30 min, 1 Hz an increased p-SMAD up to 90 min whereas mechanical stimulation at 10 Hz the effect on p-SMAD is seen up to 120 min (Fig 2 A-D).Can the authors comment on the temporality of the stimulation effect?Why do the p-SMAD levels come down at different timepoints depending on the frequency?If this is a negative feedback it must be very different from the negative feedback acting further downstream as assessed from Fig 4C .2-In the same set of experiments it is not clear how the ID1 and ID2 transcript upregulation is related to the levels of upstream p-SMAP.While the 0.03Hz stimulation does not significantly enhance SMAD phosphorylation it does give an 8fold or 3-fold increase for ID1 and ID2 transcripts, respectively.The latter effect does seem to suggest a strong BMP response and it is not clear why 0.03Hz is not taken forward for the 24h experiments.
3-Later on the occurrence of a negative feedback initiated by the long term mechanical stimulation needs to be invoked to explain the lack of a direct correlation in SMAD phosphorylation and ID1/2 transcript expression (Fig 4A and B).This is rather vague.Is there any experimental/literature evidence of what that axis might be?Does the upregulation of those negative downstream regulators also explain the lower-fold transcript levels in ID1/2 in 4-The major perturbant the authors use is acting directly on actin turnover.Instead of measuring the focal adhesions that could function as a measure of cell anchorage (FA number) and internal tension (FA number and FA size) the authors should perhaps focus on the total F-actin content.Alternatively other perturbants can be used, such as the Myosin 2 inhibitor blebbistatin (Pasapera J Cell Biol 2010).5-Collagen is used as the extracellular matrix on the 3D scaffold.While this is expected to stimulate a subset of integrins there is a change in integrin transcript levels that could be indicative of a changing extracellular matrix (Fig 2F and 4B).In particular there are increases in the transcript levels of alpha_v, beta_1 Author Rebuttal letter: Response to the reviewersâ comments We have re-worked the manuscript to address the reviewersâ concerns and we are convinced of the improved thoroughness and consistency of our data and the derived conclusions.We have highlighted all changes and amendments in the following point-by-point response.
1 Reviewer #1 o Comment 1 In this study, Gorlitz et al. explore the effect of loading frequency and duration on BMP signalling of hFOB cells within a 3D mimicking microenvironment.The rigorous investigation offers valuable insights into the mechanism governing BMP signalling.The manuscript is well presented and could be beneficial to researchers studying bone regeneration.I believe this study is acceptable for publication in Communication Biology following minor revisions to enhance its overall quality.
Line 48: Sentence does not seem correct.
Line 217 refers to Fig 53B.

Response
We thank the reviewer for the positive response.We have corrected the typos accordingly.
2 Reviewer #2 o Comment 1 It would be helpful to explain the full form of BMP-2 and its significance at the beginning of the abstract.

Response
We would like to thank the reviewer for the constructive feedback concerning our manuscript.We have revised the abstract to address the reviewersâ comment.o Comment 2 Here the authors have provided the expression levels of integrins during differential loading and compression+BMP2 simulation (B/L) (Figure 2F).It would be helpful if the authors can provide fixed sample images showing the distribution of integrins and their clustering at the focal adhesion.This can give a robust insight into integrin clustering, focal adhesion size and actin remodeling.

Response
We agree with the reviewer that the choice of phospho-paxillin for analyzing focal adhesion adaptation to load should be better explained.As paxillin is an intracellular adaptor protein localizing to focal adhesions which, similar to vinculin, it directly interacts with integrins.Phosphorylation of paxillin by focal adhesion kinase provides additional docking sites for further adapter proteins such as Src 1.We therefore chose phospho-paxillin as a marker of maturing focal adhesions which hold greater relevance for cell anchoring, integrin signaling and cytoskeletal tensioning.We have included additional histology (new Additional file 4) that illustrates the co-localization either of vinculin and phospho-paxillin or of alpha-V Integrin and phospho-paxillin.These data underline that particularly large focal adhesions as a focus of this work are enriched in any of these markers without a specific preference.Yet, phosphopaxillin provides a greater signal specificity due to the detection of the phosphorylated version.We have added the following sentence to motivate the use of phospho-paxillin for the visualization of focal adhesions: â Phospho-Paxillin represents a more selective marker of focal adhesion maturation compared to other markers such as integrins and vinculin.Yet, a strong co-localization of either of these markers with phospho-paxillin was observed, indicating that phospho-paxillin positive FAs are generally enriched in integrins and vinculin.â(lines 174-177) o Comment 3 The manuscript is well written generally, but there are a few typos that should be corrected.For example, please rephrase the sentence (line 47-48) â Bone Morphogenetic Protein 2 (BMP-2) is known to play a central role in bone development and regeneration and has gained Besides its indispensableâ ¦â .Some sentences in the manuscript can also be revisited for easier understanding.For example, the sentence "We wanted to further understand how BMP signalling responds when the time of pre-stimulation is shortened so that cells do not have time to fully adapt to the altered mechanics at all levels, and thereby find out which cellular adaptation processes are responsible for the mechanical echo and the subsequent rapid decay of the crosstalk between mechano-transduction and BMP signallingâ can be simplified to gain clarity.

Response
We have re-worked the manuscript and the particular passages indicated by the reviewer to increase clarity.

Reviewer #o Comment 1
In general the manuscript is well written but as a whole it is a bit confusing and appears to consist out of two sets of data.One displaying a message of the temporal (dis-)entanglement of the mechanical stimulus and BMP2 signalling ( Even though the authors subsequently show that Jas perturbation also disrupts loadingrelated downstream BMP signalling this does not warrant a link with focal adhesion maturation (as mentioned in the title).Perhaps the authors in this regards could make use of the benefits of their system: they could analyse/correlate single cell SMAD nuclear localisation (or phosphorylation) with number/size distribution of focal adhesions.

Response
We appreciate the reviewersâ suggestions and concerns that helped us to improve the manuscript.Despite the effect of the inhibitor on the actin cytoskeleton, the primary focus of this work was to exploit the influence of Jasplakinolide treatment on focal adhesion turnover â an effect well documented and indicated by the reviewer.The key effect of the inhibitor is the prevention of a dynamic cellular adaption in response to load.Due to the strong entanglement of cytoskeletal networks and cell adhesion clusters, the provoked alterations in the cytoskeleton also effect the focal adhesions through which cytoskeletal 82 forces are anchored to the extracellular matrix.As inhibitors acting on the cytoskeleton are well established and their mechanisms of action are known, we used Jasplakinolide in this study to alter focal adhesion remodeling and adaptation to load.We have emphasized this point in the manuscript (e.g.lines 300-302, 475-476) Concerning the very reasonable suggestion to correlate single cell Smad nuclear location to focal adhesion size, we would like to clarify that even though we attribute enhanced BMP2 signaling to focal adhesion maturation, this does not imply that the extend of Smad phosphorylation correlates with focal adhesion size on a single cell level.Our existing data underline this as we did not observe visible differences in the phospho-Smad levels of cells pre-loaded for 24h (which exhibit enlarged FAs) and non pre-stimulated cells (which show smaller and lesser FAs) (Figure 3, comparison of â câ and â L 1Hzâ ).From these data we conclude that the key element is a dynamic adaptation process of focal adhesions in response to mechanical triggers (here cyclic axial compression), not the size or maturation status of focal adhesions per se.The degree by which this focal adhesion adaptation takes place scales with the loading intensity.Hence the time window in which a beneficial effect on Smad signaling can be induced also depends on the frequency with a rather transient effect at low frequencies (1Hz) and a more persistent one at higher frequencies (10Hz) (Figure 2).Furthermore, cellular adaptation to cyclic loading cannot be expected to take place only temporally when mechanical loading is switched on, but is a continuous process of adapting to an environment featuring not only temporal alterations but also the spatial heterogeneity of cyclic straining as visible from Fig. 1C.
In parallel to this manuscript, we have submitted findings about the influence of substrate stiffness on BMP signaling, which is currently in revision at Biomaterials.In this study, we do not observe differences in BMP signaling on PDMS substrates, despite an increased FA size with increasing substrate stiffness.Yet, a very soft version of the collagen scaffold used there that permits local material deformation by cell forces in an oscillating manner, BMP signaling was also upregulated.The deformation induced by the migrating cells led to a heterogenous, non-linear deformation comparable to the material deformation resulting from the external mechanical loading applied in this study (Figure 1).Hence, we see similarities between the two situations as in both cases a dynamic environment is created in which cells, through migration, oscillate between strained and non(less)-strained regions.We believe that the dynamic iteration between differently strained regions resembles a key element that provokes a continuous cellular adaptation through FA assembly.
We have included these considerations into the manuscript at the respective passages, particularly in the discussion (lines 458-467).To avoid misunderstanding, we have further adapted the manuscript title to indicate â focal adhesion remodelingâ instead of â focal adhesion maturationâ .o Comment 2 The authors find that co-stimulation of BMP and cyclic compression enhances BMP signalling.Using 3 frequencies they find a frequency dependence: 0.03 Hz showed an increased p-SMAD in the first 30 min, 1 Hz an increased p-SMAD up to 90 min whereas mechanical stimulation at 10 Hz the effect on p-SMAD is seen up to 120 min (Fig 2 A-D).Can the authors comment on the temporality of the stimulation effect?Why do the p-SMAD levels come down at different timepoints depending on the frequency?If this is a negative feedback it must be very different from the negative feedback acting further downstream as assessed from Fig 4C.

Response
With this work, we provide evidence that the dynamic adaptation of focal adhesions is a prerequisite for the observed synergism in BMP signaling â most likely resulting from the direct interaction of BMP receptors and integrins2,3.In this context, the increase in focal adhesion maturation was frequency dependent (Figure 3).As the rate at which new focal adhesions can be formed is likely to be limited, the time until cells adopt a new steady state (and when the beneficial effect of concomitant compared to BMP-only stimulation is lost), scales with the frequency.Based on our data for Smad phosphorylation this time window is in the range of 30-90 minutes (low frequency) and up to 120 minutes (high frequency).In an attempt to achieve the original steady state (unloaded), the removal of mechanical loading leads to an internalization and retraction of focal adhesions during which BMP receptors have been observed to become internalized as well3.This explains why the removal of mechanical loading either shortly before or at the moment of BMP stimulation resulted in a less pronounced or even lacking synergism (Figure 5).All these processes are distinct from classical negative feedback loops inherent to the BMP signaling cascade such as the BMP-induced expression of extracellular antagonists (Noggin, Figure 2F) and inhibitory Smads (Smad-7, Figure 2F).Neither during concomitant stimulation, nor long term pre-stimulation these inhibitory regulators are likely to be relevant as any increase in their expression was only observed for 10Hz frequency and after 24h and can only be interpreted as the result of the significantly enhanced induction of signaling in the first place.We included these considerations in the text (see lines 213-215).o Comment 3 In the same set of experiments, it is not clear how the ID1 and ID2 transcript upregulation is related to the levels of upstream p-SMAP.While the 0.03Hz stimulation does not significantly enhance SMAD phosphorylation it does give an 8-fold or 3-fold increase for ID1 and ID2 transcripts, respectively.The latter effect does seem to suggest a strong BMP response and it is not clear why 0.03Hz is not taken forward for the 24h experiments.

Response
The regulation of gene expression by Smad proteins requires their nuclear translocation upon phosphorylation.We have recently shown that mechanical loading enhances the nuclear translocation of phosphorylated Smad proteins4.In the same study, we observed an enhanced Smad phosphorylation as early as 15 minutes after concomitant BMP stimulation and mechanical loading.Hence, an increased signaling might occur already earlier.While levels of phosphorylated Smads are only detected for the time point of analysis, gene transcript levels reflect the sum of transcription factor activity over a distinct time window where tiny differences in transcription factor activity (=Smad phosphorylation) can accumulate into more pronounced differences in the downstream signaling (gene expression).
In general, our data suggest that the level of signal amplification through the concomitant stimulation scales with the intensity (=frequency) of mechanical loading.After 24h, we no longer observed an increased expression of ID1 and ID2 between BMP stimulation and concomitant stimulation (BMP + load) for 1Hz frequency (Figure 2F).The same observation can be made for focal adhesion count and size (Figure 3), where only 10Hz frequency resulted in a visible difference.Together this suggests that, despite the beneficial effect on signal initiation, it requires a physiologically meaningful loading frequency to provoke a persistent cellular response.Due to this lack of relevance and significance, we excluded 0.03Hz from further experiments.We have included these consideration in the main text of the manuscript (see lines 140-142) o Comment 4 Later on the occurrence of a negative feedback initiated by the long term mechanical stimulation needs to be invoked to explain the lack of a direct correlation in SMAD phosphorylation and ID1/2 transcript expression (Fig 4A and B).This is rather vague.Is there any experimental/literature evidence of what that axis might be?Does the upregulation of those negative downstream regulators also explain the lower-fold transcript levels in ID1/2 in We would like to refer to a previous response (see comment 3) in which we argued that the observed transcript levels of a gene of interest can be regarded as the sum of transcription factor activity over a distinct time frame.In this regard, a lack of a direct correlation between phospho-Smad and target gene expression levels is not sufficient to speculate on active negative feedback mechanisms being initiated.We therefore do not believe that the observed differences are due to an active negative feedback mechanism.However, our data (Supplementary Figure 2) show that with 24h of mechanical loading, inhibitory Smad (Smad7) and Smad Ubiquitin Ligase (Smurf1/2) become upregulated which potentially interfere with cellular BMP signaling responses.However, for 1Hz frequency, these differences are on an overall low level and more relevant for the 10Hz frequency which we did not use for long term preloading of cells.o Comment 5 The major perturbant the authors use is acting directly on actin turnover.Instead of measuring the focal adhesions that could function as a measure of cell anchorage (FA number) and internal tension (FA number and FA size) the authors should perhaps focus on the total F-actin content.Alternatively other perturbants can be used, such as the Myosin 2 inhibitor blebbistatin (Pasapera J Cell Biol 2010).

Response
We appreciate this intriguing thought.Due to the direct coupling of the actin cytoskeleton to focal adhesions it is practically impossible to selectively interfere with actin turnover but not focal adhesion formation and vice versa.As suggested, we have performed additional experiments using the cell force inhibitor blebbistatin that led to a strong reduction in focal adhesion count and size.Furthermore, this treatment not only reduced the basal BMP signaling, but also prevented any load-induced synergism.
Together, these new data underline our conclusion that the increase in the BMP signaling response is linked to a dynamic focal adhesion remodeling and maturation process.Although acting primarily on the cytoskeleton, the advantage of Jasplakinolide is that it does not directly lead to a disassembly of the FAs as observed for blebbistatin.Both inhibitors hereby interfere with the load-induced synergism which can be linked to the potential to form and remodel focal adhesions.The fact that by using blebbistatin focal adhesion size decreases to a non-physiological level is in line with previous reports in which a non-physiological cell-rounding due to inefficient adhesion on very soft substrates (<1kPa) leads to integrin internalization and ultimately reduced BMP signaling3.We have added this consideration into the discussion part of the main manuscript (see lines 409-412).
o Comment 6 Collagen is used as the extracellular matrix on the 3D scaffold.While this is expected to stimulate a subset of integrins there is a change in integrin transcript levels that could be indicative of a changing extracellular matrix (Fig 2F and 4B).In particular there are increases in the transcript levels of alpha_v, beta_1

Response
We agree with the reviewer that the question of how cells anchor within the here used biomaterial niche is an important point â particularly when focusing on integrins.We have included additional histology that demonstrates the adsorption of fibronectin on the collagen walls of the biomaterial scaffold.This fibronectin originates from the bovine serum which is added as a supplement to the cell culture medium (new Additional file 3) and forms a thin layer directly during cell seeding.In contrast to the culture of primary human fibroblasts in the scaffold5, this laver is mostly unchanged by the cell line used in this study throughout the pre-incubation time (2 days prior to stimulation).Particularly, no fibronectin fibers were found in the scaffold as they are typically deposited by fibroblasts5 or MSCs.We furthermore stained selectively for human collagen type I and type IV as important fibrillar respectively networkforming ECM proteins.Together, these new data indicate that the hFOBs used in this study are adhering to a mixture of the original collagen-I material, a thin adsorbed fibronectin layer which remains mostly stable throughout culture and a minor deposition of collagen type IV by hFOBs over 24h of culture.We therefore assume that changes in recognized adhesion patterns are negligible and cannot explain differences in BMP2 signaling between conditions representing different culture durations.
We have added the following passage to the main text (lines 413-419): â Changes in the composition of the extracellular matrix can provoke alterations in the involvement of different integrin subtypes in focal adhesions potentially further influencing BMP-2 signaling.Immunhistology of relevant ECM proteins however showed that the culture of hFOBs in the biomaterial scaffold did not alter the presence of collagen-I and fibronectin and only mildly increased the presence of collagen IV (Additional File 3).Consequently, cell-secreted ECM and resulting changes in ECM composition are not expected to play a relevant role in the described BMP-2 signaling response to cyclic loading.âo Comment 7 Is the memory (mechanical echo) frequency dependent?Response With this study, we provide evidence that a dynamic adaptation process of focal adhesions is linked to an increased BMP signaling response.As our data indicate that the degree by which this adaptation process takes place is frequency-dependent, we speculate that the corresponding time window, here termed â mechanical echoâ , is likely to increase with the intensity of the mechanical stimulus.We have included this aspect in the discussion section (see lines 375-378).o Comment 8 Version 1: Reviewer comments: Reviewer #3 (Remarks to the Author) In their response Gorlitz et al have clarified all of my concerns.Therefore, I support the publication of the current manuscript.
The authors have elucidated their choice of correlating focal adhesions with the changes in BMP signalling upon inhibition of the actin turnover using Jasplakinolide.Indeed, the strong entanglement between the tension in cytoskeletal networks and the adhesion complex remodelling makes independent measurements challenging.The authors have now addressed this connection clearly at several places in their manuscript and further strengthened the importance of an adaptation of the focal adhesions to the cyclic stimuli as opposed to a maturation per se which seems key to their data and interpretation.The agreement between Jas treatment, myosin inhibition (blebbistatin experiments shared in the response) and cells on soft substrates in terms of the reduced load-induced synergism with BMP signalling is very intriguing.In fact, the comment of similarity of the data presented here and to cells migrating on a non-linearly deformed soft collagen scaffold that undergo temporal changes by interacting with differently strained regions is very interesting.Presumably the oscillating focal adhesion turnover dynamics is what drives increased BMP signalling as opposed to uniform substrates (even if at different stiffness).The mechanical echo described in this manuscript might be long enough for the cells to cross a soft collagen patch and keep BMP signalling augmented.
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Fig 2, 4 and 5) and a second discussing focal adhesions (Fig 3 and 6).Their relation is not entirely clear.Focal adhesions are as expected bigger in situations where forces are applied to cells (one of many examples is Riveline J Cell Biol 2001).Disrupting actin turnover using Jas directly affects loading-related focal adhesion growth (Galbraith Science 2007, Parsons Nat Rev Mol Cell Biol 2010).
Fig 2F (compared to Fig 2E: 90 min versus 24hrs, respectively)?Response , and beta_3 indicative of RGD containing ECM (Hynes Cell 2002).Would a change in ECM chemistry change the BMP signalling response?Could this explain the difference summarized in Fig 5, for example between [24h L + 90â B; 98% crosstalk] and [90â L pre B; 50% crosstalk]?